This proposal is designed to examine some aspects of the mechanisms which control corticotropin (ACTH) secretion by cells of the anterior pituitary gland. The functional differentiated mouse pituitary tumor cell strain AtT 20/D16v has retained the ability to secrete ACTH and to respond to a variety of controlling signals for 15 years in continuous culture. These cells will therefore be used to study individual inhibitory and stimulatory components of the ACTH regulatory system. The independent manipulation of various control signals and the study of their interrelationship are difficult or impossible to perform in the intact mammalian organism. Evidence for the existence of an autofeedback mechanism in AtT 20/D16v cells has already been obtained: exogenous ACTH added to the cells inhibits endogenous hormone secretion while removal of secreted ACTH by frequent medium changes stimulates hormone secretion. I wish to extend these observations to determine if the feedback activity of ACTH on its own production by AtT 20/D16v cells is mediated by specific membrane receptors (analogous to ACTH receptors of adrenocortical target cell membranes) and if receptor binding is associated with modulation of intracellular cyclic AMP concentration. These experiments will involve the binding of radioactively labeled ACTH to both intact cells and to isolate membrane preparations. Since AtT 20/D16v cells produce a variety of ACTH molecules differing in size and biological activity, I plan to examine the effectiveness of the various molecular species of tumor cell ACTH as well as of a variety of synthetic ACTH analogs in the negative feedback regulation. Newly synthesized radioactively lablled ACTH0will be isolated by immunoprecipitation in order to establish whether the autofeedback control involves altered rates of hormone synthesis.